A synthesis of the external morphology of cypridiform larvae of Facetotecta (crustacea: Thecostraca) and the limits of the genus Hansenocaris

Abstract Although the naupliar and cypridiform stages of the enigmatic y‐larvae of Facetotecta have been found in the marine plankton worldwide, they still represent the last significant group of crustaceans for which the adult forms are still unknown. From a number of y‐cyprids representing different taxa from different locations, we employ scanning electron microscopy to describe fine morphological details of all external structures of this unique larval form. We document different segmentation patterns of the abdomen and presence/absence of the labrum and structural differences in the antennules, labrum, paraocular process, thoracopods, and telson lend support for the erection of several new genera as opposed to the single Hansenocaris. The data presented here emphasize the morphological limits of the genus Hansenocaris and the “bauplan” of cyprydiform larvae of Facetotecta. Although the optimum pathway is a joint analysis of both molecular and morphological characters, we use the morphological characters of y‐cyprids to align them cladistically and determine the limits of the genus Hansenocaris s.s. and describe common characters for all y‐cyprids including six pairs of the lattice organs instead five pairs considered as a ground pattern for all Thecostraca. We also determine plesiomorphic and apomorphic characters of all known y‐cyprids and separate them from other thecostracan cypridiform larvae.


| INTRODUC TI ON
Although the naupliar and cypridiform stages of the enigmatic ylarvae of Facetotecta have been found in the marine plankton worldwide, they still represent the last significant group of crustaceans for which the adult forms are still unknown (Glenner et al., 2008;Grygier, 1996;Høeg et al., 2014;Kolbasov et al., 2021a;. Different facetotectan nauplii were first described in detail more than 100 years ago by Hansen (1899), who originally illustrated five different naupliar types of y-larvae from West Indian, equatorial Atlantic waters, and from the Bay of Kiel in the Baltic. Subsequently, y-larvae were reported from almost all oceans in the world (Belmonte, 2005;Kolbasov et al., 2021a;Ponomarenko & Korn, 2006). A post-naupliar instar or "y-cyprid" (Figure 1), resembling other thecostracan cypridiform larvae, was first described by Bresciani (1965). Treatment with the crustacean molting hormone 20-hydroxy ecdysone has been shown to induce y-cyprids to molt into a unique minute, slug-like stage, called the ypsigon (Glenner et al., 2008). The morphology of both the y-cyprid and the ypsigon suggest that unknown adult stages are advanced endoparasites in still to be identified hosts (Glenner et al., 2008;Pérez-Losada et al., 2009). Thus, the incompletely known life cycle of Facetotecta includes free-swimming naupliar stages, a cypridiform larva specialized for attachment and an ypsigon with an unknown role (Høeg et al., 2014;Pérez-Losada et al., 2009). The y-nauplii are either planktotrophic (feeding) or lecithotrophic (nonfeeding), but the y-cyprid is always nonfeeding.
At least thirteen naupliar morphotypes are known to date, but only some of these have been correlated with y-cyprids. This not only challenges adequate taxonomic classification but it also highlights a considerable knowledge gap on the lifecycle of y-larvae and the structural variation in y-cyprids.
The naupliar body consists of a cephalic anterior part, covered by the dorsal head shield, and a posterior part or hindbody. The ycyprid has a univalved carapace that only partially covers the larval body, six pairs of natatory thoracopods, a segmented thorax, and a limbless abdomen terminating with a telson with furcal rami ( Figure 1). The dorsal side of the naupliar head shield, the "trunk," F I G U R E 1 Diversity of y-cypris larvae of Facetotecta, general view, lateral side (SEM). (a) H. itoi from arctic White Sea, subtidal zone, thoracic segments numbered in Roman, abdominal segments numbered in Arabic. (b) Y-cypris larva from boreal Kuril-Kamchatka trench, abyssal zone, depth 3000-5640 m. (c) H. papillata from equatorial Indonesia, subtidal zone. (d) H. spiridonovi from subtropical Azores Islands, subtidal zone. a1-antennule; fr-furcal rami; lb-labrum; te-telson; thp1-6-thoracopods 1-6. Scale bars in μm. the carapace, and the telson of the y-cyprid have a surface pattern of reticulated cuticular ridges, which together form a series of interconnected plates or "facets".
A recent study revealed seven naupliar instars in Hansenocaris itoi , instead of the five that were previously supposed for the Facetotecta (Kolbasov et al., 2021b). This number of naupliar instars is unique not only for Facetotecta but also for Thecostraca and Hexanauplia as well. Itô and Takenaka (1988), Itô (1989), Grygier (1987), Høeg and Kolbasov (2002), Kolbasov et al. (2007) and Kolbasov, Savchenko, and Høeg (2021) studied various aspects of the external and internal morphology of cyrpidoform larvae of Facetotecta in detail and discussed their relationships with other crustaceans.
Two informal morphological groups of facetotectan y-cyprids were recognized by Kolbasov and Høeg (2003). The first, the "Hansenocaris pacifica group," includes y-cyprids with a long carapace with a round anterior end and sharp, laterally elongated posterior margins, and curved antennular hooks. This group includes all the Atlantic y-cyprids, H. itoi, and also H. pacifica representing a type species of genus Hansenocaris, H. furcifera, and probably H. papillata. Y-cyprids of the other group have a shorter head shield, often with an elongate and sharp anterior end, and supposedly lacking curved antennular hooks. Hansenocaris rostrata, H. acutifrons, and H. tentaculata belong to this group. This latter grouping has hardly any taxonomic value because of the very distinct morphology of H. tentaculata (e.g., the two-segmented abdomen, instead of a foursegmented one) compared to other Facetotecta.
Our studies of different y-cyprids also revealed that some forms have labrum with numerous small spines instead of five long spines or lacking labrum at all (own unpublished data). These facts (different segmentation of abdomen, presence/absence of labrum, different morphology of antennules, labrum, paraocular process, thoracopods, and telson) indicate on the presence of several separate genera instead a single Hansenocaris. Thus, the known morphological variation of y-cyprids challenges the concept of Hansenocaris and begs the question what the limits of the genus and the "bauplan" of y-cyprids is?
Here, we describe in detail the morphology of six y-cyprids from Kamchatka, Russia (Figure 2), and supplement with data on the morphology of other species and specimens studied by us (Figure 1).
These y-cyprids from Kamchatka belong to the "Hansenocaris pacifica"-group, and may represent at least three different species.
We systematically employ scanning electron microscopy to describe the morphology of all external structures of y-cyprids. We reveal several morphological features that separate the "true" genus Hansenocaris from other representatives of Facetotecta and describe common characters for all y-cyprids including six pairs of the lattice organs instead five pairs, which is presently considered as a ground pattern for all Thecostraca.

| MATERIAL AND ME THODS
The material was obtained in 2019 during the survey of the plankton collections of Zoological Institute RAS (St.-Petersburg) and included six specimens of different y-cyprids ( Figure 2)  We compare our SEM data obtained for cypridiform larvae to those from other locations: H. itoi from the White Sea (see , H. papillata from coastal waters off Indonesia (see Kolbasov et al., 2007), H. spiridonovi from Azores (see Kolbasov et al., 2021a), and two undescribed species from Taiwan and abyssal Kuril-Kamchatka Trench.

| Morphological characters and taxonomical prospectus
In this study, we examined the following morphological characters (see also Table 1).

| Carapace and its structures (Figures 1-5 and 11, Table 1)
The carapace of all y-cyprids is univalved and resembles an inverted boat hull and only partially covers the larval body (Figures 1, 2 and 3). In most of the y-cyprids studied, the cuticle of the carapace is not densely length of studied y-cyprids from Kamchatka varies from 560 to 648 μm and from 440 to 470 μm for their carapaces, with a carapace to length ratio at 0.64 to 0.8 (see Table 1 for other Facetotecta). The anterior end of the carapace in most studied y-cyprids, including all from the "Hansenocaris pacifica"-group, is rounded (

| Antennules (Figure 6)
The antennules of y-cyprids, including described and undescribed species, seem always to consist of four segments, these being rather similar in all studied species and specimens (Figure 6a,b,f).
The large, first segment may actually consist of several fused ones (Bresciani, 1965;Schram, 1970). Such fusion is also argued for the basal segment in cirripede cyprids . The first segment has external cuticular folds and resembles the basal antennular segments in other thecostracan cyprids in lacking any armament ( Figure 6a,b,f). The second segment is horseshoe shaped and resembles the attachment third segment of the antennule in cirripede cyprids. In many y-cyprids, this segment (Table 1), including all from the "Hansenocaris pacifica"-group, is armed with a conspicuous curved hook ("claw") at the distal margin, putatively serving for host attachment ( Figure 6). This claw is significantly larger than the second segment in y-cypris from the Deep Kuril-Kamchatka Trench, while it is comparable (smaller) with this segment in other y-cyprids ( Figure 6f,g). Three species (H. acutifrons, H. rostrata, and H. tentaculata) lack this claw altogether ( Table 1). The minute lateral seta presents on the outer surface of second segment (Figure 6g). This seta  TA B L E 1 (Continued) was firstly described by Grygier (1987) and afterward we found it in all SEM-examined larvae. The third, short segment bears one lanceolate seta and one small seta on the distal margin (Figure 6f,g).
Since these species were studied with a light microscopy only, some details may have been missed or misinterpreted. The fourth segment is small and armed terminally with one long, distally serrated seta, one very short seta and one "thorn" probably representing a rudimentary seta (Figure 6f,g). Subterminally, it also carries a long aesthetasc ( Figure 6). The form of aesthetasc is different: it may be narrow, ribbon shaped (Table 1; Figure 6c,d) or having bulbous proximal and distal parts separated by a very characteristic constriction (Table 1; Figure 6a,b,e-g). For H. rostrata and H. acutifrons, Itô described in the fourth segment only two setae (short and long), while a subbasal aesthetasc was indicated for the third segment, probably erroneously (Itô, 1984b(Itô, , 1985. (Table 1; Figures 6a,b and 7b-f)

| Labrum
The majority of y-cyprids possess a prominent labrum consisting of the wider basal/proximal part and a more or less bulbous distal part (Figure 7b-d). The labrum is reduced to a blunt swelling in H. acutifrons (Itô, 1985) or entirely absent in y-cypris from the Deep Kuril-Kamchatka Trench ( Table 1).
The wider basal part of the labrum has a wrinkled cuticle and covers a slit with a mouth (Figure 7d). This part bears a pair of big papilliform lateral pores with a cuticular rim (Figure 7c,d).

| Paraocular process and postocular filamentary tuft
The paraocular processes and postocular filamentary tufts are located laterally to the bases of antennules (Figures 6a and 7a). Often these structures are hidden by the margin of carapace, but they are found in all facetotectan species and also in unidentified y-cyprids (see Table 1). Thus, their presence likely represents the ground pattern feature for all Facetotecta. The paraocular processes are bifurcated and connected by a basal part with the co-lateral compound eye (Figure 7a). Both position and shape suggest that they represent the external portion of the organs of Bellonci (Itô & Takenaka, 1988), therefore being homologous to the frontal filaments and glendronach of other thecostracan larvae (Walker, 1974;Høeg et al., 2003; own unpublished TEM images). The paraocular process consists of a swollen basal part and narrow bifurcate distal part terminating with anterior and posterior protrusions or rami (Figures 6a,b and 7a,g). Normally these rami are equal and not strongly elongated, and the paraocular process is shorter than the antennule. Only in H. tentaculata the process is longer than the antennule, and with unequal length rami (Itô, 1986; Table 1).
A pair of postocular filamentary tufts is situated posteriorly to the paraocular processes ( Figure 7a). Each tuft consists of a proximal cylindrical stalk that is normally hidden by the margin of carapace ( Figure 7a) and a distal part with 9-15 setiform protrusions (Figures 6b and 7a,c,d). It was shown by Itô and Takenaka (1988) that these tufts have a secretory nature. Figures 1, 2 and 8) The thorax consists of six segments (thoracomeres) bearing biramous appendages (thoracopods). Thoracomeres 2-6 have tergites with serrate posterior margins, and the last two thoracomeres have pleural extensions with different degree of development ( Figures 1, 2 and 8a,b). The first thoracomere has no individual tergite and is distinct only ventrally (Figure 8e). Based on thoracopod 1 muscle insertions, Grygier (1987) supposed that first two thoracomeres are dorsally fused, although this supposition needs to be proved by further TEM studies as a fusion of thoracomere 1 with the head is also possible. Each tergite is also equipped with two or three transverse and several short longitudinal cuticular ridges (Figures 1 and 8a,b). In all y-cyprids, the last two thora-  1a, 2d,e and 8b). The lateral articulate membrane covering thoracopod insertions bears four irregular proximal sclerites or "coxicules" (Grygier, 1987;Itô, 1989; Figure 8a-  in terminology of Itô, 1989) is inserted between the basal coxal sclerites and does not connect with the thoracopod coxae; two shorter and wider plates lie anteriorly and posteriorly.

| Thorax and thoracopods (
Each thoracopod (Figure 8) consists of a basal array of sclerites, a coxa, a basis, and a pair of rami (exopod and endopod). Except where the inner seta in the middle part marks the merging of the two distal segments. A conspicuous constriction is often present at the merging site of these two distal segments. 1; Figures 1,  2, 9 and 10) In all y-cyprids, except H. tentaculata, the abdomen consists of three short segments and a long telson with two furcal rami ( Figures 1, 2 and 9). In H. tentaculata, the abdomen encompasses F I G U R E 4 Anterior lattice organs of y-cypris larvae of Facetotecta (a, e-y-cypris larva 2; b, d-y-cypris larva 4; c-y-cypris larva 3; f-y-cypris larva 6. SEM). Lattice organs indicated by magenta color in b-e. (a, b) Locations of anterior lattice organs (lo1-3) on carapace, anteriormost unpaired pore with cuticular rim indicated by asterisk. (c, d) Locations of lattice organs 2 and 3. (e, f) Lattice organ 1. bp-big central unpaired pores; lo1-3-lattice organs; pc-pore with cuticular rim; ps-pit/pore with seta inside; tp-terminal pore of lattice organ. Scale bars in μm.

| Abdomen and furcal rami (Table
only a single short segment and a long telson (Itô, 1986). The  Table 1). Tergites of abdominal segments may bear pores/ pits with or without seta inside (Figure 9b), while the ventral surfaces have a pair of small, rounded pores at the bases of pleural extensions (Figure 9c). The midventral part of the first abdominal segment has a small and bifurcated outgrowth (Figures 8f   and 9c) interpreted as a putative penis rudiment (Grygier, 1987;Itô, 1989).
The telson is densely covered by chitinous serrate cuticular ridges, forming dorsal, lateral, and ventral longitudinal rows of plates, having normally more or less rectangular shape and symmetrical pattern that is sometimes broken towards the posterior end (Figures 1, 2 and 9a-f). In the majority of y-cyprids, the telson has two dorsal rows of cuticular plates; each lateral side bears two rows, laterodorsal and lateroventral; the ventral surface ordinary consists of five indistinct, longitudinal rows of plates (one central, two ventrolateral, and two ventromedial). The telson in H.
tentaculata has only two irregular rows of dorsal plates, one row of elongated lateral plates, and no distinct plates on ventral side (Itô, 1986).  Table 1).
A pair of short furcal rami is inserted at the posterior end of the telson (Figures 1a, 9a,b,e and 10). Each ramus resembles a twoannulated structure due to the more or less developed circular cuticular ridge (Table 1; Figure 10c,d), but it is in fact unsegmented (unjoined). The furcal rami of almost all described species and studied y-cyprids carry three wide, lanceolate setae of different lengths, with serrate margins (Figure 10b, nos 2-4, Table 1), but H. acutifrons differs in possessing only two such setae (Itô, 1985). In all y-cyprids studied with SEM, we found a tiny distal seta with terminal pore (no 1) and an outer basal papilla with pore (Figure 10b-d).

| Morphological similarities and differences between y-cyprids of Facetotecta
The diversity of the Facetotecta is seen best in the y-cypris.  (Kolbasov et al., 2008;Kolbasov, 2009; own data). The presence of such ornamentations seems not to be correlated with cypris size and their putative adaptational function is completely obscure. While cirripede cyprids have F I G U R E 6 Cephalic appendages and structures of y-cypris larvae of Facetotecta (a-y-cypris larva 1; b-y-cypris larva 5; c-y-cypris larva 6; d-y-cypris larva 3; e, g-y-cypris larva 2; f-y-cypris larva 4. SEM). (a) Anterior part of y-cypris exuvium, ventral side (ypsigon exit opening in anterior end indicated by asterisk). (b) Anterior part, lateroventral view (paraocular process colored in red; thin and long seta of 4th antennular segment indicated by arrowhead). (c-e) distal parts of antennules (narrow, ribbon-shaped aesthetascs without constriction couloured in red in 'C' and 'D'; segments numbered in Arabic in 'C'). (f) Antennule (segments, setae and hook indicated by different colors; segments numbered in Arabic, setae indicated by arrowheads).

(a)
(c) a highly reduced abdomen with short telson (Kolbasov et al., 1999), the ascothoracid larvae possess developed 4-5-segmented abdomen with long telson (Kolbasov et al., 2008; own data), although their telson lacks cuticular ridges forming plates, is flattened laterally and bears only a pair of telsonic spines (in contrast to normally 4-6 telsonic spines in y-cyprids, see Table 1). All six thoracomeres in cypridiform larvae of Ascothoracida and Cirripedia are separated but the tergite of first thoracomere in y-cyprids is fused with the cephalon or with the second thoracomere in Grygier's (1987) inter- The cirripede cyprids also possess four-segmented antennules, but they lack a curved hook comparable to that in y-cyprids, and their third segment bears an attachment disc covered with cuticilar villi and having exit pores for both the multicellular cement gland and unicellular glands (Bielecki et al., 2009;Høeg et al., 2003). Thus four-segmented antennules of Facetotecta are unique and not homologous to four-segmented antennules of other thecostracan cypridiform larvae (see also Grygier, 1987). On the other hand, it is very likely that the fourth segment of cirripede cyprids evolved by fusion of the two distal segments in y-cyprids. Itô and Takenaka (1988) established that the paraocular process is connected with the compound eye and thus putatively homologous to the frontal filaments of other thecostracan larvae, but its bifurcate shape is unique within Thecostraca. The postocular filamentary tufts are characteristic only for y-cyprids and absent in other thecostracans. They may therefore represent an autapomorphy for the taxon.

| Lattice organs
Although the previous characters were already known for y-cyprids, the presence of six instead five pairs of the lattice organs is described here for the first time. The presence of five (two anterior and three posterior) pairs of the lattice organs was seen as a fundamental symplesiomorphic character of the carapace for all Thecostraca (Høeg & Kolbasov, 2002;Jensen et al., 1994). The lattice organs were subsequently described in Cirripedia, Ascothoracida, and Facetotecta  (Elfimov, 1986;Høeg & Kolbasov, 2002;Itô & Grygier, 1990;Jensen et al., 1994). The lattice organs have a similar anatomy throughout and are homologous in all Thecostraca. TEM reveals that they are chemoreceptors and evolved originally from free setae (Høeg et al., 1998).  (Hanstrøm, 1947) and one species of Decapoda (Laverack & Sinclair, 1994), and there is no information about the innervation of posterior dorsal organ and the lattice organs.
Høeg and Kolbasov (2002)  pace. If the first thoracomere fuses with cephalon forming cephalothorax (5 + 1), instead with second thoracomere, as Grygier (1987) suggested, this could explain the presence of six pairs of the lattice organs in y-cyprids. In parasitic Tantulocarida, which may belong to Thecostraca (see Petrunina et al., 2014), the free-swimming males possess a carapace resembling that in y-cyprids of Facetotecta (Petrunina & Kolbasov, 2012). This carapace bears seven pairs of big pores/pits with a tuft of sensillae inside that are likely homologous to the lattice organs, although this is pending ultrastructural investigation. Interestingly, the tantulocaridan males also possess a cephalothorax that incorporates the two first thoracomeres (5 + 2). This may explain the presence of seven pairs of sensillate pores. Rybakov et al. (2003) showed that the lattice organs in cyprids correspond to setae in the preceding nauplius, thus ontogeny also emphasizes the setal origin of these fascinating structures in thecostracan cypridiform larvae.

| Phylogeny
Although several groups of y-cyprids were proposed on the basis of their morphology (see Introduction here; Kolbasov et al., 2008), there is no cladistic analysis to formalize the relations between described facetotectan species and unspecified y-cyprids. As a pioneering attempt, we here use the characters of y-cyprids to align them cladistically and de- incorporate comprehensive and specimen-based character resolution for their y-cyprids. Therefore, we conduct a morphological analysis and argue that the character matrix here set up will be of much future value, even if the topology of the tree will undoubtedly see future changes.
For all described species of Facetotecta and SEM studied ycyprids we developed a matrix of 15 characters for the Nexus Data Editor 5.0 ( Table 2). Data were scored "0" or "1," when both conditions were present (we avoided multistate data), "-" for inapplicable states, and "?" for unknown state. Eight characters (numbers 1, 2, 3, F I G U R E 9 Abdomen of y-cypris larvae of Facetotecta (a-y-cypris larva 6; b-y-cypris larva 3; c-y-cypris larva 1; d, g-y-cypris larva 2; e-y-cypris larva 5; f-y-cypris larva 4. SEM  These data were subjected to parsimony analysis and a search of the shortest trees (PAUP 4.0, Swofford, 1998). All characters were entered unordered and of equal weight, and all trees were unrooted.
We reconstructed bootstrap 50% majority-rule consensus and neighbor-joining trees ( Figure 12). These trees show that species H.  process and is unique within Facetotecta. We suppose that each of these species forms separate taxa (genera at least). On the other hand, we presently abstain from formal taxonomic steps. This is best done on a much larger collection of species, where at least a considerable number are also characterized by molecular markers.

| Summary and outlook
One of the most provoking results of our study is the presence in y-cyprids of six instead five pairs of the lattice organs. The pres- (iii) form of antennular aesthetasc; (iv) development of labrum; (v) size of rami in paraocular process; (vi) segmentation of thoracopods; (vii) segmentation, armament and ornamentation of abdomen and (viii) setation of furcal rami. These differences evidence on the presence of several separate genera instead of a single Hansenocaris.
The characters reviewed and discussed here will be useful for future phylogenetic efforts, in which species are grouped on both molecular and morphological characters.
The genus Hansenocaris s.s. is characterized by (i) more or less elongated carapace with rounded anterior end and developed cuticular ridges at least at anterior part; (ii) developed labrum with five long spines; (iii) antennules with hook smaller or comparable with second segment; (iv) paraocular process with equal rami and shorter than antennules; and (v) abdomen four segmented; (vi) telson with serrate spines along posterioventral margin.
F I G U R E 1 2 Cladistic reconstruction cladogram of the species and y-cyprids of Facetotecta (all characters unordered and of equal weight; PAUP, Swofford, 1998; parsimony-informative characters indicated in brackets): (a) Bootstrap 50% majority-rule consensus tree (node corresponding to "H. pacifica -group" indicated by star). Percentages at nodes denote frequency of occurrence among 100 trees. (b) Neighbor-joining tree.

ACK N OWLED G M ENTS
We thank the collaborators of the Laboratory of Electronic Microscopy of Moscow State University for assistance in SEM studies. We are obliged to anonymous reviewers for their comments and criticism.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All morphological data will be upload to Dryrad upon acceptance.
SEM stubs are stored in the Kolbasov lab at the Moscow State University and can provide for examination upon on request.